Poster given for the Cellular Imaging for Biological Research and Drug Development meeting (Cambridge Healthtech Institute, 12-14 November 1998, SanDiego) Multi-photon (nP) excitation of fluorescence is based on the principle that multiple photons can interact "simultaneously" with a fluorophore and thereby add their energies to excite a transition with multiple times the energy of each individual photon (Denk 1990; Williams 1994; Denk 1995; Xu 1996; Xu 1996). Serotonin absorption bands exist at wavelengths less than 250 nm; thus 2P and 3P absorptions are expected at ~ 500 and ~700 nm respectively. After optical excitation, a serotonin molecule relaxes vibrationally through environmental interactions and releases a fluorescent photon with an energy less than that of the total energy absorbed (~340 nm). In these imaging studies we utilize low energy red photons (740 nm) and a three photon mechanism to activate native serotonin fluorescence. Red light 3P excitation alleviates the need for deep UV optics and their associated aberrations and low transmissivities. Nonlinear excitation is also intrinsically localized to the focal plane and thus is potentially less invasive than single-photon techniques. We have previously shown that 3P microscopy of serotonin-granules in RBL-2H3 cells is possible (Maiti 1997). Here we report 3P imaging measurements of those cells undergoing antigen stimulated secretion. Sampling at one frame every 10-20 seconds, we observe each vesicle's contents to disappear suddenly with no evidence for granule motion. Single granule release rates can be determined and indicate release that is spatially coupled and sometimes oscillatory. Upon exodus granules often leave behind clearly defined footprints, suggesting undissolved serotonin still bound to the granular matrix. Denk, W., D. W. Piston and W. W. Webb (1995). Two-photon molecular excitation in laser-scanning microscopy. Handbook of Biological Confocal Microscopy. J. B. Pawley. New York, Plenum Press: 445-458. Denk, W., J. H. Strickler and W. W. Webb (1990). "Two-photon laser scanning Maiti, S., J. B. Shear, R. M. Williams, W. R. Zipfel and W. W. Webb (1997). "Measuring serotonin distribution in live cells with three-photon excitation" Science 275:530-532. Williams, R. M., D. W. Piston and W. W. Webb (1994). "Two-photon molecular excitation provides intrinsic 3-dimensional resolution for laser-based microscopy and microphotochemistry." FASEB J. 8: 804-813. Xu, C. and W. W. Webb (1996). "Measurement of two-photon excitation cross sections of molecular fluorophores with data from 690 to 1050 nm." J. Opt. Soc. Am. B 13(3): 481-491. Xu, C., et al. (1996). "Multiphoton fluorescence excitaiton: new spectral windows for biological nonlinear microscopy." Proc. Natl. Acad. Sci. 93: 10763-10768.